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Abstract:

Described is an intra-mammary teat sealant and a corresponding method of
forming a physical barrier in the teat canal of a non-human animal for
prophylactic treatment of mammary disorders during the animal's dry
period. The method includes the step of infusing a teat seal formulation
including a metal salt in a glyceride-containing gel base into the teat
canal of the animal. The method also prevents the formation of black spot
defect in dairy products, especially cheddar cheese, made from the milk
of animals so treated.

Claims:

1. A method of forming a physical barrier in the teat canal of a non-human
animal for prophylactic treatment of mammary disorders during the
animal's dry period and simultaneously preventing black spot defect in
dairy products made with milk from the animal, the method
comprising:infusing an amount of a teat seal formulation into the teat
canal of the animal, wherein the teat seal formulation comprises a
non-toxic metal salt, dispersed in a gel phase comprising a
glyceride,wherein the amount of the teat seal formulation infused is
sufficient to form a physical barrier to entry of microorganisms into the
teat canal, and wherein the teat seal formulation does not cause black
spot defect in diary products made with milk from the animal.

2. The method of claim 1, comprising infusing a teat seal formulation that
is devoid of anti-infective agents.

3. The method of claim 1, comprising infusing a teat seal formulation that
is devoid of bismuth.

4. The method of claim 1, comprising infusing a teat seal formulation that
is devoid of anti-infective agents and devoid of bismuth.

15. The teat sealant of claim 12, wherein the teat sealant is devoid of
anti-infective agents.

16. The teat sealant of claim 12, wherein the teat sealant is devoid of
bismuth.

17. The teat sealant of claim 12, wherein the teat sealant is devoid of
anti-infective agents and devoid of bismuth.

18. The teat sealant of claim 12, comprising at least about 30% by weight
of the non-toxic metal salt.

19. The teat sealant of claim 12, comprising about 50% to about 75% by
weight of the non-toxic metal salt.

20. The teat sealant of claim 12, comprising about 65% by weight of the
non-toxic metal salt.

21. The teat sealant of claim 12, wherein the non-toxic metal salt is
selected from the group consisting of titanium salts, zinc salts, barium
salts, and combinations thereof.

22. The teat sealant of claim 12, wherein the non-toxic metal salt is
selected from the group consisting of titanium dioxide, zinc oxide,
barium sulfate, and combinations thereof.

23. In a method of forming a physical barrier in the teat canal of a
non-human animal for prophylactic treatment of mammary disorders during
the animal's dry period, the method comprising the step of infusing a
seal formulation into the teat canal of the animal, an improvement
comprising:infusing a teat seal formulation as recited in claim 12.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]Priority is claimed to provisional application Ser. No. 61/207,879,
filed Apr. 8, 2009, the content of which is incorporated herein by
reference. This application is also related to co-pending application
Ser. No. 11/869,966, filed Oct. 10, 2007, which is incorporated herein by
reference.

FIELD OF THE INVENTION

[0002]The invention is directed to a metal-containing, intra-mammary teat
sealant to prevent mastitis in dry cows. The intra-mammary teat sealant
does not cause visual defects in dairy foods (especially cheese) made
from milk from treated animals. The invention is further directed to a
method to prevent "black spot defect" (BSD) in cheese.

[0004]Because of the difficulty in treating mastitis, prevention of new
intra-mammary infections is a major focus in the dairy industry. The rate
of new infections is significantly higher during the dry period as
compared to new infections during the lactating period. (For example, one
study showed that 61% of all new gram-negative intra-mammary infections
occurred during the dry period. See Todhunter et al. (1995) J. Dairy Sci.
78:2366.) The three-week period immediately following dry off, and the
two weeks prior to calving, are periods particularly prone to new
infections. Thus, in recent years dairy producers have focused a
considerable amount of effort in "preventive maintenance" of cows during
their dry period.

[0005]In April of 2003, an internal (or "intra-mammary") teat sealant
(ITS) for use in dry cows was introduced in the US market. Marketed in
the US under the "ORBESEAL" trademark (US Trademark Registration Nos.
2,772,198 and 3,120,693), the product was developed in New Zealand. The
"ORBESEAL"-brand ITS introduced into the US market contains 65% w/w
bismuth sub-nitrate dispersed in a viscous paste. The ITS product does
not contain any antibiotics, nor does the product contain any active
antimicrobial agents. The ITS is injected into the teat end using a
tubular applicator syringe, in the same fashion as applying a dry cow
antibiotic. The ITS product fills the fissures and folds of the teat
canal, thereby creating a physical barrier to pathogens. See U.S. Pat.
No. 6,254,881, issued Jul. 3, 2001, incorporated herein by reference.

[0006]Initial studies of the "ORBESEAL"-brand product in New Zealand
concluded that the product functioned as well as a broad spectrum,
long-acting intra-mammary antibiotic in preventing new intra-mammary
infections at calving and in preventing clinical appearance of mastitis
through the first five (5) months of lactation. See Woolford et al.
(1998) New Zealand Veterinary Journal 46:1. A more recent study in the US
also concluded that this ITS product improved the udder health of cows
already infused with cloxacillin benzathine. See Godden et al. (2003) J.
Dairy Sci. 86:3899-3911. Thus, the "ORBESEAL"-brand ITS has proven to be
an effective tool in reducing the number of new cases of mastitis in
dairy cows during their dry period. Despite its relatively recent
introduction into the US market, the "ORBESEAL"-brand product has enjoyed
widespread market acceptance and is used extensively in US dairy herds.
In short, the "ORBESEAL"-branded product is very good for its intended
purpose of preventing mastitis.

[0007]Subsequent to the introduction of the "ORBESEAL"-brand ITS product
in the US, a visual defect in aged dairy products, most notably aged
cheddar cheeses, began to appear. The visual defect takes the form of
small, black spots (roughly 0.5 to 5 mm in diameter) that appear
throughout the aged cheese. The spots are a purely aesthetic, visual
defect that lowers the graded quality (and hence the market value) of the
cheese affected with the problem. The spots are not accompanied by any
organoleptic defect in the cheese. Cheese affected with the black spots
is saleable, albeit at a lower grade than unaffected cheeses. The defect
has been termed "black spot defect" (BSD).

SUMMARY OF THE INVENTION

[0008]A first version of the invention is directed to a method of forming
a physical barrier in the teat canal of a non-human animal for
prophylactic treatment of mammary disorders during the animal's dry
period and simultaneously preventing BSD in dairy products made with the
animal's milk. The method comprises infusing a teat seal formulation into
the teat canal of the animal. The teat seal formulation may contain
bismuth or may be bismuth-free. The formulation is administered in an
amount sufficient to form a physical barrier to entry of microorganisms
into the teat, but does not cause black spot defect in diary products
made with milk from the animal. Preferably, the teat sealant is devoid of
anti-infective agents (i.e., the teat sealant preferably does not contain
antibiotics or other anti-infective active agents). Preferably the method
comprises infusing a teat seal formulation comprising at least about 30%
by weight of a non-toxic metal salt, more preferably about 50% to about
75% by weight of the non-toxic metal salt, and more preferably still
about 65% by weight of the non-toxic metal salt. The purpose of the salt
is primarily to impart sufficient density to the composition so that the
ITS "settles" into the teat canal.

[0009]In one version of the method, the non-toxic metal salt is selected
from the group consisting of bismuth salts, titanium salts, zinc salts,
barium salts, and combinations of these salts. The most preferred salts
are bismuth sub-nitrate, titanium dioxide, zinc oxide, barium sulfate and
combinations of these salts. Other non-toxic salts of these metal are
explicitly within the scope of the invention, such as halides, sulfates,
phosphates, carbonates, nitrates, sulphamates, acetates, citrates,
lactates, tartrates, malonates, oxalates, salicylates, propionates,
succinates, fumarates, maleates, and the like.

[0011]Thus, in its most preferred form, the intra-mammary teat sealant
comprises, in combination, a gel base comprising a glyceride; and a
non-toxic metal salt dispersed in the gel base. The preferred
corresponding method thus comprises infusing an amount of a teat seal
formulation into the teat canal of the animal. The teat seal formulation
comprises a non-toxic metal salt dispersed in a gel phase comprising a
glyceride, wherein the amount of the teat seal formulation infused is
sufficient to form a physical barrier to entry of microorganisms into the
teat canal. The teat seal formulation also does not cause black spot
defect in diary products made with milk from the animal.

[0012]Another version of the invention is directed to an intra-mammary
teat sealant consisting essentially of a gel base including mono-, di-,
and/or triglycerides, in combination with a non-toxic metal salt
dispersed in the gel base, wherein the metal salt is bismuth sub-nitrate.
As noted earlier, the teat sealant preferably comprises at least about
30% by weight, more preferably about 50% to about 75% by weight, and more
preferably still about 65% by weight of the metal salt.

[0013]Yet another version of the invention is an improvement to
intra-mammary teat sealants. Specifically, in a method of forming a
physical barrier in the teat canal of a non-human animal for prophylactic
treatment of mammary disorders during the animal's dry period, wherein
the method comprises the step of infusing a seal formulation into the
teat canal of the animal without an anti-infective agent, the improvement
of the present invention comprises infusing a teat seal formulation
comprising a non-toxic metal salt(s) in a glyceride-containing gel base.
The improvement prevents the formation of black spot defect in dairy
products made from the milk of treated animals.

[0014]Numerical ranges as used herein are intended to include every number
and subset of numbers contained within that range, whether specifically
disclosed or not. Further, these numerical ranges should be construed as
providing support for a claim directed to any number or subset of numbers
in that range. For example, a disclosure of from 1 to 10 should be
construed as supporting a range of from 2 to 8, from 3 to 7, 5, 6, from 1
to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

[0015]All references to singular characteristics or limitations of the
present invention shall include the corresponding plural characteristic
or limitation, and vice-versa, unless otherwise specified or clearly
implied to the contrary by the context in which the reference is made.

[0016]All combinations of method or process steps as used herein can be
performed in any order, unless otherwise specified or clearly implied to
the contrary by the context in which the referenced combination is made.

[0017]The methods of the present invention can comprise, consist of, or
consist essentially of the essential elements and limitations of the
methods and products described herein, as well as any additional or
optional ingredients, components, or limitations described herein or
otherwise useful in synthetic organic chemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIGS. 1A and 1B are photographs of a typical black spot defect in an
18 kg block of aged white cheddar cheese. FIG. 1A shows the surface of
the block, with a black spot defect readily visible. FIG. 1B is a
magnified view (with a superimposed ruler) showing the dimensions of the
defect. Such spots are equally distributed throughout the cheese.

[0019]FIG. 2 is an electron photomicrograph of a black spot defect
exhibiting characteristic hair- or rod-like structures of bismuth (III)
sulfide nanorods. No such structures appeared in any non-BSD cheeses or
non-BSD cheese regions tested. The rods shown in FIG. 2 have diameters
ranging from 37.09 nm to 129.33 nm.

[0020]FIG. 3 is an electron photomicrograph of a single rod-like structure
from a black spot defect region. The rod shown in 130.88 nm in diameter
and exhibits a line bisecting the length of the rod.

[0021]FIGS. 4A and 4B are images of laboratory-induced black spot defects.
In FIG. 4A, the various components of "ORBESEAL"-brand intra-mammary teat
sealant (ITS) were blended with cheese and each spot was photographed
immediately. FIG. 4B shows the same spots photographed after exposure
either to volatiles from aged cheddar cheese or hydrogen sulfide gas.
Sites 4 and 5 contain bismuth subnitrate and the intact "ORBESEAL"-brand
ITS formulation, respectively.

[0022]FIG. 5 is a histogram depicting the cleanability of various types of
ITS's.

[0023]FIG. 6 is a graph depicting viscosity of the ITS formulations at
different temperatures. X-axis depicts temperature in ° C., Y-axis
depicts viscosity.

DETAILED DESCRIPTION OF THE INVENTION

[0024]Starting in late 2003, a number of inquiries have been made by
cheese makers to the University of Wisconsin-Madison, Department of Food
Science and Center for Dairy Research (CDR), seeking information about
the appearance of a novel "black spot defect" in aged cheeses, notably
aged cheddar cheese. Historically, grey to black discolorations in cheese
have been the result of several different and distinct causes, including
the growth of specific microorganisms (e.g., certain environmental
propionibacteria or molds) or the contamination of cheese with food-grade
lubricant debris. The particular BSD noted by the cheese makers, however,
did not fit the profile of a bacterial contaminant or other spoilage
organism, nor did it appear to be lubricant debris that had found its way
into the milk stream or other debris introduced during the cheese-making
process.

[0025]Thus, the first step was to determine the chemical structure of the
black spot defect. A great deal of effort was initially made to extract
the affected regions of BSD cheese. Extraction efforts using a wide
spectrum of organic solvents of varying polarity, hydrophobicity, etc.,
proved fruitless as a means of isolating any type of organic pigment mass
from the cheese matrix. Although extraction of the BSD with organic
solvents was not successful, the extraction efforts did yield useful
data. Notably, because the pigment did not dissolve or diffuse into such
solvents, it could be concluded (with a high degree of probability) that
the black spot pigment likewise would not dissolve or diffuse within the
cheese matrix itself.

[0026]Visual examination of a growing number of cheese samples exhibiting
the defect (samples accumulated from commercial cheese makers) confirmed
this conclusion--the black spot pigment is well contained and does not
appear to diffuse into the cheese matrix. See FIGS. 1A and 1B, which are
photographs of a typical 18 kg block of aged, white cheddar cheese
affected with the BSD. FIG. 1A is a photograph of the outer surface of
the cheese block. FIG. 1B is a magnified view of a single black spot,
with a ruler superimposed on the image to show the dimensions of the
spot. Spots such as the one shown in FIGS. 1A and 1B typically are
equally distributed throughout the cheese block, ranging in size from
<1 mm to about 5 mm in diameter. The frequency of the spots within any
given 18 kg cheese block varies widely, from <10 per block to well
over 100.

[0027]There were some anecdotal reports received from cheese makers that
specific aging and storage strategies might aid in dissolving or
diffusing the spots to the point that they are no longer visually
noticeable. (Because the defect is not accompanied by any organoleptic
deficiency, "fading" the spots would ameliorate the condition.) Such an
effect, however, is highly unlikely given the stability of the pigment to
the organic solvents employed in the extraction efforts. In short, the
extraction experiments performed by the present inventor used solvents
having hydrophobicities similar to milk fat. If the black spot pigment
dissolved or diffused into the cheese matrix itself (via an aging or
storage protocol), the pigment should likewise readily dissolve or
diffuse into an organic solvent having physical characteristics similar
to milk fat. That result did not occur in the lab. Moreover, given the
typical pH/acidic environment in cheese, and the typical aging/shelf life
periods associated with most aged cheddar-type cheeses (0.5 to 2 years),
the anecdotal evidence that the defect can be ameliorated via aging or
storage protocols is without merit.

[0028]One experiment, however, proved most enlightening: the black spot
pigment is readily dissolved in nitric acid. This strongly suggested that
the pigment was an inorganic salt. Coupled with the timing of the first
appearance of the defect, a working hypothesis was formulated, namely
that the ITS was either a causative agent of (or at least correlated
with) the BSD. The discovery that the black spot pigment readily
dissolves in acid supported a further hypothesis that the pigment may be
comprised of bismuth III sulfide. Thus, it was concluded the
"ORBESEAL"-brand product, which in the US contains 65% by weight of a
bismuth-containing salt, was likely being inadvertently introduced into
the milk stream. As noted above, the "ORBESEAL"-brand product has been
commercially successful because it forms a tight physically barrier to
the entry of pathogens into the teat canal. However, removing the product
from a treated animal requires stripping of the animal's teats. It
appeared that some of the ITS remained in the teats after stripping and
was finding its way into the cheese milk.

[0029]The next phase of research operated pursuant to a hypothesis that
bismuth III sulfide was in fact the causative agent of the BSD. Bismuth
subnitrate itself is white and relatively chemically inert. Thus, its
trace presence in fluid milk, mozzarella cheese, and yogurt is not
readily apparent visually. However, in aged cheeses with high flavor
intensity, the black spot defect appears prominently. Thus, it was
hypothesized that bismuth III sulfide (a black, relatively insoluble
salt) was the product of a reaction between bismuth subnitrate (from the
ITS) and hydrogen sulfide produced within the aging cheese by the actions
of ripening microflora, enzymes, and certain cofactors acting on the
protein/amino acid components of cheese.

[0030]In short, the hypothesis was that bismuth subnitrate made its way
into the milk stream due to incomplete removal of the ITS prior to
milking. The bismuth subnitrate then reacted with hydrogen sulfide to
yield bismuth III sulfide according to Equation 1

[0031]In addition to having a specific elemental target, bismuth, it was
hypothesized that, under the conditions or chemical environment present
within the cheese matrix, the Bi2S3 molecules would form a
crystalline structure referred to in the literature as nanorods or
nanowhiskers. See W. Zhang et al. (2001) Sol. State Comm. 119:143-146 and
B. Zhang et al. (2006) J. Phys. Chem. 110:8978-8985. These
bismuth-containing nanorods would thus constitute light-diffracting
particles capable of imparting the grey to black hue seen in the black
spot defect.

[0032]Efforts were then focused on confirming: 1) the elemental presence
of bismuth in the black spot defects; and 2) confirming the physical
presence of bismuth III sulfide nanorod structures within the black spot
defects.

[0033]Confirming the presence of bismuth within the black spots was
investigated using inductively coupled plasma mass spectroscopy (ICPMS).
AOAC International (Association of Analytical Communities) method 993.14
was used. The first efforts screened multiple black spots for the
presence of several elements that could be contributing to BSD. The
initial experiments focused on metal salts/oxides typical of those found
in milk- and cheese-handling/conveying equipment, and other residual
metal derivatives present in food-grade processing. As a measure of
control, cheese compositional analyses were conducted. Specifically,
protein, ash, and moisture were measured using methods 2001.14, 935.42,
and 926.08, of the Official Methods of Analysis, AOAC 17th Edition,
respectively (copyright 2000, ISBN: 0935584-67-6). Fat was measured
according to the method described in the Official Methods of Analysis,
AOAC 17th Edition.

[0034]Transmission electron microscopy (TEM) studies were performed as
follows: approximately 100 μl double-distilled water was added to
samples and the mixture was pulverized into a suspension with a glass
rod. Approximately 5 μl aliquots of suspended sample were deposited
onto polyvinyl alcohol-formaldehyde acetal-coated 300 mesh copper TEM
grids (Ted Pella, Inc., Redding, Calif.). Excess sample was wicked away
with small sections of filter paper and the remaining sample was dried to
the surface of the grid at room temperature. In some cases, NANO-W-brand
TEM negative stain (Nanoprobes, Incorporated, Yaphank, N.Y.) was applied
over the dried sample to enhance contrast and visibility. Specimens were
observed with a Philips CM 120 electron microscope and images were
collected with a MegaView 3 Digital camera (from SIS, Ringoes, N.J.).
Measurements were taken with SIS-brand analysis software (Ringoes, N.J.)
calibrated with reference samples of known lengths.

[0035]ICPMS results demonstrated the presence of the elements chromium,
copper, iron, nickel, and bismuth in the BSD region. Although incremental
increases in the elements chromium, copper, iron, and nickel were found,
bismuth concentrations in the BSD region were routinely three orders of
magnitude greater than the same cheese assayed in non-BSD areas. These
results show that bismuth is the only element present in sufficient
quantities to participate in a pigment-generating reaction.

[0036]Several hundred TEM images of BSD regions of cheese samples were
captured with a single, consistent conclusion. Nanorods typical of those
reported in the literature cited above were uniquely present in the BSD
cheese region. An example of such an image is presented in FIG. 2. The
nanorod structures shown in FIG. 2 are too small to be readily detected
with a light microscope. The nanorods shown in FIG. 2 range in diameter
from about 69 nm to about 130 nm. The nanorods are very stable to the
potentially abusive conditions of TEM. The rods appear to have a slightly
mottled surface and they exhibit a characteristic line running the length
of the nanorod. See FIG. 3, which is an increased magnification view of a
single nanorod. The presence of such structures is consistent with the
presence of bismuth sulfide nanorods formed under the conditions present
in the cheese matrix.

[0037]To confirm the reactivity of bismuth subnitrate as a reactant in
forming bismuth III sulfide nanorods, additional assays were conducted to
see if BSD could be purposefully recreated in the lab. In short, cheeses
were manufactured with known amounts of ITS components and subjected to
either the authentic volatile gasses produced by maturing cheeses or
exposed directly to the hypothesized bismuth subnitrate co-reactant,
hydrogen sulfide gas. In both situations, the responses were invariably
the same: When cheese samples containing bismuth subnitrate or the
complete ITS formulation were exposed to authentic cheese volatiles or to
"chemical standard"-grade H2S gas, each formed identical black
pigmentation with the accompanying presence of nano-rod structures,
further confirming that bismuth subnitrate is the culprit in BSD. The
results are shown in FIG. 4. No other ITS component formed black spots
when so exposed. Furthermore, the susceptible sites formed identically
black pigmentation when exposed to either authentic cheese volatiles or
to hydrogen sulfide gas, thus confirming that hydrogen sulfide gas was
the suspected co-reactant.

[0038]From a cheese manufacturing and aging or ripening standpoint, it is
not reasonable to consider targeting the elimination of hydrogen sulfide
gas production as a means of controlling BSD. Hydrogen sulfide is a
highly aroma active compound, the product of microbial, enzymatic and
co-factor activities against sulfur-containing amino acids such as
cysteine. See Arfi et al., (2002) Appl. Microbiol. Biotechnol.
58:503-510.

[0039]There is ample research to support the claim that hydrogen sulfide
gas is a necessary and/or valued component of typical aged cheddar cheese
flavor. See Burbank & Qian (2005) J. Chrom. 1066:149-157. Even if a
scheme was devised to eliminate the production of hydrogen sulfide (by
interrupting of dozens of complex metabolic pathways) the resulting final
product runs the risk of a flavor character unacceptable to cheese
graders and consumers.

EXAMPLES

[0040]The following Examples are included solely to provide a more
complete description of the invention disclosed and claimed herein. The
Examples do not limit the invention in any way.

Example 1

[0041]A test ITS using a combination of zinc oxide and titanium oxide as
the metal salts was formulated. The test ITS was identical to the
"ORBESEAL"-brand formulation, with the exception that it did not contain
any bismuth or bismuth-containing salts. The test ITS comprised zinc
oxide, titanium dioxide, mineral oil (30-40%), and aluminium stearate.

[0042]To prepare a batch of ITS, liquid paraffin (e.g., mineral oil) is
delivered into a suitable vessel equipped with a mixer. Aluminum stearate
is added and the mixture is stirred and heated to about 160° C.
until homogeneous (about two hours). The non-toxic, non-bismuth
containing salt is then added in portions to the mixture, with stirring,
until the desired amount of metal salt has been added. The mixture is
then stirred until homogenous. The products is then transferred into
conventional injector tubes for intra-teat administration.

Example 2

[0043]The object of this Example was to compare retention within the teats
of non-lactating dairy cows of an ITS that did not include bismuth
sub-nitrate as compared to the "ORBESEAL"-brand product.

[0044]The study was performed at the Blaine Dairy of the University of
Wisconsin-Madison (UW), in Arlington, Wis. Sixteen (16) cows (n=64 teats)
were enrolled on the day of dry off. All enrolled cows were required to
have four functional quarters and no visible sign of mastitis. All cows
were dried off and received intramammary antibiotic dry cow therapy (DCT)
according to standard UW dairy herd protocols. Parity and milk yield (at
dry off) were recorded for each cow. Upon initial enrolment, teats were
scored for shape, length, diameter and degree of teat end hyperkeratosis.
Within each cow, two teats were assigned to receive the "ORBESEAL"-brand
ITS and two teats were assigned to receive the test ITS. The
administration protocol was designed to ensure that each product was
administered uniformly among teat locations, eight teats each per product
administered in each location (right-rear, right-front, left-rear,
left-front). Sealant tubes were weighed before and after administration
to determine the net volume administered. Prior to receiving DCT & the
internal teat sealant, teat ends were cleaned using a single 70%
isopropanol alcohol wipe and partial insertion technique was used to
reduce the probability of introducing teat skin pathogens. After
administration of the internal sealant, teats were dipped with an
external teat disinfectant.

[0045]Teats were examined on Days 1, 2, 3, 4, 5, 6, 7, 14, 28, 42 and at
calving to detect redness, swelling and/or sealant leakage. On Days 14,
28, 42 and at calving, sealant was removed from one teat (eight teats for
each sealant per removal day) of each cow by hand stripping. The removed
sealant was collected with the first milk into graduated 50 ml plastic
vials. The vials were centrifuged (3000 rpm×5-7 minutes), the
supernatant rinsed, and the recovered sealant weighed. The amount of
recovered sealant was compared at each period between the test
ITS-treated teats and the "ORBESEAL"-brand ITS-treated teats. Follow up
samples were collected at Day 1 post-calving using the same procedure. At
all sampling periods, after removal of the sealant, teats were dipped
with an external teat disinfectant. After calving, quarter milk samples
were aseptically collected from all quarters and cultured to identify
intramammary infections.

[0046]Group Characteristics--Teat Length and Volume:

[0047]A total of 16 cows were enrolled into the study for a total number
of 64 teats; 32 teats received the test ITS and 32 teats received the
"ORBESEAL"-brand ITS. The teat length and volume for the test population
is shown in Table 1:

[0048]There was no significant difference in teat length or volume for
teats in Group A or Group B (p>0.36). Overall teat length was 5.11 cm,
ranging from 3.3 cm to 7.3 cm. The average teat length was 5.12 cm for
Group A and 5.11 cm for Group B, and ranged from 3.3 cm to 7.3 cm for
Group A and from 3.5 cm to 7.10 cm for Group B. A two Sample paired
t-test was performed to test the null hypothesis that the mean teat
length in the two treatment group did not differ. There was no
significant difference in teat length between teats randomized to receive
either product (p=0.95).

[0049]The overall teat volume was 24.66 cm3, ranging from 12.47
cm3 to 54.34 cm3 (std. dev. 9.17 cm3). The mean teat
volume was 23.6 cm3, ranging from 12.54 cm3 to 54.34 cm3
(std. dev. 8.60 cm3) in Group A. In Group B, mean teat volume was
25.71 cm3, ranging from 12.47 cm3 to 48.25 cm3 (std. dev.
9.73 cm3). A two sample paired t-test was used to test the null
hypothesis that the teat volume in the two groups did not differ. There
was no significant difference in teat volume between teats randomized to
receive either product (p=0.95 and p=0.35; log transformed analysis).

[0050]Hyperkeratosis: Teat-end health was scored for hyperkeratosis using
the following scale: No ring (N), Smooth Ring (S), Rough (R), Very Rough
(VR). The distribution of teat scores was: N (n=21; 32.8%), S (n=31;
48.4%), R (n=11; 17.2%) and VR (n=1; 2%). An X2 test confirmed that
the distribution of hyperkeratosis score was not associated with
treatment group (p=0.13).

[0051]Amount of Sealant Administered, Recovered and Lost: Statistical
analyses using a paired t-test were performed to determine if the amount
of sealant administered, recovered, or lost (not recovered) did not
differ based on treatment group.

[0052]Of the four (4) grams in each tube, the overall amount of sealant
administered was 3.62 grams. There was no significant difference in the
amount of "ORBESEAL"-brand ITS (3.46 gram) or test ITS (3.62)
administered (P=0.12).

[0053]Overall, the amount of sealant recovered was 0.82 gram and there
were no significant differences based on treatment (P=0.89). The overall
amount of sealant lost was 2.88 grams and did not differ by treatment
group (P=0.40). The amount of sealant recovered tended to be associated
with recovery date (P=0.08) with more sealant recovered on day 14 as
compared to other recovery periods (day 14, recovery=1.6 grams; day 28
recovery=0.68 grams; day 42 recovery=0.65 grams; calving recovery=0.33
grams).

[0054]Simple linear regression was used to determine that there was no
significant relationship between the amount of administered sealant and
teat volume (p=0.59, p=0.53).

[0055]For the recovered sealant a simple linear regression test was
performed to test the null hypothesis that there was no significant
linear relationship between the amount of recovered sealant and the teat
volume. Only 6% of the recovered sealant was accounted for by teat volume
(P=0.05).

[0056]The proportion of administered sealant was not significantly
associated with teat volume, while the recovered sealant was correlated
significantly with the teat volume but only for a small proportion (6%).

[0057]A one-way ANOVA was used to determine univariate relationships
between the amount of administered and recovered sealant and the teat
position, the product, the cow.

[0058]The object of this Example was to develop an ITS with improved
cleanability properties. In this Example, an ITS similar to the one
described in Example 1 was made, but the mineral oil gel was replaced
with a triglyceride-based mobile phase. These sealants, which are
preferred, proved to have good barrier properties, as well as improved
cleanability properties using conventional teat-cleaning protocols (as
compared to mineral oil-based sealants). Milk producers have found that
it is extremely difficult to remove residual from conventional, mineral
oil-based ITS formulations from milk contact surfaces using standard
cleaned-in-place (CIP) procedures. Therefore, an ITS which has good
barrier properties and which is also easy to clean from surfaces that
contact milk is very desirable in the market.

[0059]Due to various factors, many milk producers do not adequately remove
ITS from the udder before milking. It is unlikely that this outcome can
be eliminated through training and education efforts. Furthermore, many
milk producers do not routinely achieve the recommended temperature and
cleaning compound concentration requirements during clean up, thus
exacerbating the build-up of residue from conventional ITS formulations
on milking equipment. Dairy producers' cleaning practices are not likely
to change through education efforts. However, a viable approach to
improving the cleanability of residual ITS resides lies in altering the
chemistry of the ITS product itself.

[0060]The salt component of the ITS, whether it be bismuth sub-nitrate,
zinc oxide or some other salt, is not influenced or solubilized to any
great degree using standard cleaning protocols. The salts themselves are
dispersed particulate matter. The salts are not soluble in either the
continuous gel phase (conventionally mineral oil), nor in the aqueous
cleaning phase. Further still, the mineral oil component (a viscous
mixture of alkanes) of an ITS product is essentially non-reactive to
standard cleaning chemicals (e.g. chlorinated alkaline cleaner). Mineral
oil is also sufficiently non-polar to resist solubilization by aqueous
cleaning solution. In short, once an ITS comprising a mineral oil gel
phase is deposited onto a milk contact surface, it is extremely difficult
to remove the ITS using standard cleaning protocols.

[0061]The role that mineral oil plays in an ITS formulation is as an inert
continuous phase dispersant. The present inventor has discovered that
this role can also be played by materials such as a mono-, di-, and/or
triglyceride oil. The benefit of replacing mineral oil with, for example,
a triglyceride material, is that unlike mineral oil, triglyceride is
reactive to the most common milk equipment cleaning agent, alkaline
cleaner (i.e., sodium hydroxide). Fatty acids esterified as glycerides
are chemically converted at high pH with sodium hydroxide into their
corresponding sodium salts, e.g. sodium stearate, and are readily removed
from surfaces and dispersed into the aqueous phase of the cleaning
solution. Triglyceride oils such as corn, cottonseed or canola oil are
readily available, are heat stable (for sterilization purposes), and can
be altered to have different stability and melting properties.

[0062]Glyceride-based ITS's compare very favorably with mineral oil-based
ITS's in terms of barrier properties. The triglyceride-based ITS's also
exhibit at least a 10-fold improvement in cleanability as compared to
mineral oil-based ITS's. Because of the improved cleanability, a
triglyceride-based ITS may utilize a bismuth-containing salt, such as
bismuth sub-nitrate as the salt component.

[0063]To test ITS's made from a glyceride gel base, a small clean-in-place
(CIP) loop was constructed. The CIP loop comprised an 11-gallon tank,
flexible sanitary lines, a centrifugal pump, a throttling valve, and a
digital flow meter. For test surfaces, 2''×2'' coupons of 316
stainless steel and plastic were used. The cleaned coupons were weighed.
Samples of known weight of ITS were then applied to the surface of each
coupon and the coupons weighed again. The coupons were then washed in the
CIP loop. Each coupon was inserted into the end of one of the flexible
lines of the CIP lop and held in place with a hose clamp. The pump was
then turned on and the flow rate modulated with the throttling valve to
achieve the desired target flow rate as displayed on the digital flow
meter. A digital thermocouple was used to monitor the temperature. With
the coupon and ITS treatment in place, the system was filled with hot
water at 130° F., the pump turned on and adjusted for flow. One
(1) oz of chlorinated alkaline cleaner per gallon of water in the CIP
loop was added and the system was allowed to operate for 10 minutes. The
coupon was removed, dried and weighed to determine the amount of ITS
removed.

[0064]The results demonstrate that there are no unique differences in
cleanability between the "ORBESEAL"-brand ITS and the zinc-based ITS
described in Example 1, both of which use mineral oil as the gel phase.
See Table 5, shown in FIG. 5, n=3. Table 5 depicts the weight percent
removal of ITS treatments from stainless steel and plastic coupons. Key:
ss=stainless steel surface; pl=plastic surface; orb="ORBESEAL"-brand ITS;
zmo=zinc+mineral oil; zcot=zinc+cottonseed oil; zcan=zinc+canola oil.

[0065]In contrast, ITS formulations made with a triglyceride as the gel
phase, rather than mineral oil, resulted in a dramatic (e.g., 10-fold)
increase in the degree to which the residual soil is removed. See FIG. 5.
Without being limited to a specific mechanism, it is believed that the
improved cleanability is a result of the increased chemical reactivity of
triglyceride as compared to the non-reactive mineral oil. In short, the
ester bond of the triglyceride material is readily hydrolyzed by the
basic cleaning agent (NaOH). As such, the soil is dispersed into the
aqueous phase as a sodium salt of the corresponding fatty acids.

Example 4

Dairy Product Reactivity

[0066]Fluid milk samples (2% homogenized, vitamin D) were inoculated with
0.05 wt % of each ITS treatment and stored at 45° F. for two
weeks. Afterward, the samples were visually evaluated for signs of
spoilage using sensory methods using an expert panel (n=4). There were
four ITS treatments evaluated, including "ORBESEAL"-brand ITS, Zn/mineral
oil, Zn/canola oil, and a control sample. The panelists were asked to use
a difference from control assessment. In short, the panelists were asked
to evaluate the control sample, then rate the treated samples on flavor
with the control milk as a reference. Results were as follows:

[0067]There were no visual signs of protein destabilization whatsoever.
Each of the treated milk samples appeared undistinguishable from the
control. Flavor assessments yielded similar results in that no
significant flavor defects or attributes arose as a function of the
presence or type of ITS treatment.

[0068]Cheese analysis is still in progress. Freshly made mozzarella and
cheddar cheese samples were manufactured and the surfaces were treated
with ITS, vacuum packaged, and are currently in an aging program. To
date, no deleterious signs or appearance defects are present.

Example 5

Yogurt Fermentation and Sensory Assessment

[0069]Three different yogurt fermentations were conducted in order to
investigate a possible effect of the presence of the ZnO sealant or the
"ORBESEAL"-brand ITS on the final sensory characteristics of this
fermented product. In previous experiments, it was determined that the
presence of such materials did not alter the drop in the pH of the
product in a noticeably way. Hence, the following experiment was
performed: 50 mL of pasteurized milk were place in three sterile 100 mL
flasks, and inoculated with 0.5 mL of a suspension containing
Lactobacillus delbrukeii spp. bulgaricus (in Elliker's broth, previously
incubated for 24 hour at 37° C.). 50 mg of "ORBESEAL"-brand ITS
was added to one of the flasks and 50 mg of the ZnO sealant to other (65%
in heavy mineral oil), the third flask was used as the control treatment.
The flasks were incubated overnight at 37° C. and assessed for
differences in the final pH, as well as flavor and aroma profiles.

[0070]Results: The final pH of the three fermented products was very
similar, and ranged between 4.45 and 4.5. No differences in flavor and
aroma profiles were detected between the three treatments when tasting
the final products.

Example 6

Density of ITS Formulations

[0071]The densities of 7 different zinc-based ITS formulations were
determined and compared to "ORBESEAL"-brand ITS. Aliquots (400 μL) of
each of the sealant preparations were weighed using an analytical scale.
Table 6 summarizes the results obtained for each sample. The tabular
entries correspond to the average of at least 10 measurements with
corresponding standard deviations. In general, the density of the
suspensions increased when using higher amounts of ZnO. The performance
criterion was a range of 5-10% of the density of the original
"ORBESEAL"-brand ITS. This is a range of from about 1.817 to about 2.009
g/mL at the 5% level and about 1.722 to about 2.104 g/mL at the 10%
level. All of the samples tested fell within these parameters with the
exception of the 60% ZnO Canola Oil treatment at the 5% level.

[0072]The viscosities of newly prepared ITS comprising zinc oxide,
aluminum stearate, and canola oil was determined, at several
temperatures, using a Brookfield Digital Viscometer Model DV-I Prime
(Brookfield, Middleboro, Mass.) fitted with a S06 Spindle at 2.0 rpm. The
effect of the temperature on the viscosity of these new ITS formulations
can be observed in FIG. 6. Data at lower temperatures (-15° C.) is
in progress.